A. Field of the Invention
This invention relates to an apparatus, a kit and method for processing ova and parasites from human and animal specimens. More specifically, this invention relates to a multi-filter apparatus, kit, and method for separating and concentrating ova and parasite from human and animal specimens.
B. Description of Related Art
The prompt diagnosis and treatment of parasitic infections in humans and animals requires simple, fast and accurate diagnostic techniques. Parasitic infections can be diagnosed based on the presence of ova and parasites in human or animal specimens. For example, parasitic infections in the human intestine can be diagnosed by the presence of ova and parasites in a patient's fecal material. Diagnoses based on the presence of ova and parasites are faster because these parasitic forms are more likely to be present in such specimens. The ability to detect ova and parasites is increased when the ova and parasites are separated from residual host material. The separated ova and parasites can then be qualitatively and quantitatively analyzed to determine the nature and extent of the infection.
There is a need for a separation apparatus which quantitatively and non-selectively separates ova and parasites from human or animal specimens while minimizing the amount of residual contaminating material. Such residual material can interfere with subsequent identification and analysis of the collected parasite sample. There is also a need for a separation apparatus which does not require the use of toxic separation chemicals. Prior separation devices typically use toxic chemicals such as ethyl acetate and ether. Finally, prior separation devices typically offer a compromise between efficiency and cost. The present invention overcomes the deficiencies of prior devices by providing a highly efficient, yet inexpensive, multi-filter separation apparatus which minimizes the use of toxic separation chemicals.
One of the traditional methods for collecting ova and parasites is the floatation method. In this method, a specimen, such as fecal material, is dispersed in a flotation solution. The ova and parasites are then collected by adjusting the specific gravity of the solution so that ova and parasites float to the surface, where they are collected on a microscope slide.
A U.S. Patent to R. J. Greenwald (U.S. Pat. No. 4,293,405) describes an apparatus for collecting ova using the floatation method. This patent is incorporated by reference herein in its entirety. In the Greenwald apparatus, a fecal specimen, suspended in a buoyant solution, is placed in a lower tray, and then a single filter is placed over the specimen. Because the filter pore size is greater than 500 micrometers, the filter allows the ova to pass upwards through the filter and collect on the surface of the solution.
A U.S. Patent to T. Addis (U.S. Pat. No. 3,905,895) discloses a similar apparatus and method for separating ova from fecal material. This patent is incorporated by reference herein in its entirety. The Addis apparatus includes a hollow receptacle and a pair of interconnected filtering surfaces. Fecal samples are placed within the receptacle, a flotation solution is added, and the ova are allowed to pass through the filters to the surface of the solution, where they are collected.
A disadvantage of both the Greenwald and Addis devices is that the ova are concentrated at the liquid meniscus, and not in a tight pellet. The large pore size of the filters in the Greenwald and Addis devices also allows contaminating matter to collect at the liquid surface with the ova. This residual material interferes with analysis of the parasite samples.
Another method for separating ova and parasites from fecal material is filtration prior to a gravimetric centrifugation separation. In this method, the sample is first filtered through a filter to remove large contaminants. Then, diethyl ether is added to "defat" the specimen solution. After separation by centrifugation, 4 layers were formed: an ether layer, a debris layer, a formalin layer and a pellet containing ova and parasites. After decanting the first three layers, the pellet was analyzed for the presence of ova and parasites. A later improvement in this method substituted ethyl acetate for diethyl ether to reduce the risk of fire or explosion.
A U.S. Patent to W. S. Zierdt (U.S. Pat. No. 4,081,356) discloses an apparatus and method for collecting ova and parasites using the traditional formalin ether method. This patent is incorporated by reference herein in its entirety. The Zierdt apparatus includes a single filter juxtaposed between emulsification and separation chambers. Emulsified fecal samples are transferred from the emulsification chamber to the separation chamber by vertical agitation. After transfer of the sample to the emulsification chamber, the ova and parasites are subsequently concentrated using the formalin-ether method.
A disadvantage of the Zierdt apparatus is that the pore size of the single filter must be sufficiently large to allow transfer of the parasite sample through the filter using vertical agitation. Typically, only large residual material is trapped by the filter. Vertical agitation can also generate fine sediment which contaminates the parasite sample. The Zierdt apparatus is also designed for use of hazardous chemicals such as diethyl ether, which create a fire and explosion hazard.
A U.S. Patent to M.-G. Chang (U.S. Pat. No. 5,066,463) discloses a multi-purpose fecal examination apparatus for performing occult blood tests as well as inspecting fecal samples for parasites. This patent is incorporated by reference in its entirety. Fecal samples are inserted into a hollow tube within the apparatus. Filter paper impregnated with test reagent may be placed in contact with the sample within two windows in the apparatus. After testing is complete, the fecal samples are drained through a single filter into a collection device such as a test tube. Because the examination apparatus lacks a self-contained collection tube, the use of this apparatus increases the likelihood that fecal samples will be lost or will contaminate the user. Because the filtration apparatus includes only a single filter, and is designed to collect the sample by draining the sample through a filter, the apparatus requires a large pore size and allows residual material to collect with the parasite sample.
Perry et al. reviewed the effectiveness of five different filtration devices, including the Zierdt apparatus described above (Perry, J. L., Matthews, J. S., and Miller, G. R. "Parasite Detection Efficiencies for Five Stool Concentration Systems." Journal of Clinical Microbiology, vol. 28, no.2 (June 1990), pp. 1094-1097). This article is incorporated by reference in its entirety. These filtration devices were used for the removal of large debris before a traditional centrifugation concentration procedure, such as the formalin-ether or formalin-ethyl-acetate methods. The devices have large filter pore sizes ranging from 600 to 2,000 micrometers. While all tested devices detected all six test organisms, the efficiencies of detection were generally low.
Other methods of analyzing specimens for the presence of parasites utilize non-filtration techniques. For example, U.S. Patents to H. Adlercreutz et al. (U.S. Pat. No. 4,427,769) and M. A. Grow and V. D. Shah (U.S. Pat. No. 5,094,956) disclose methods for testing fecal samples for hemoglobin by immunoassay testing. These patents are incorporated by reference herein in their entirety. Such tests are not designed to facilitate visual detection or identification of ova and parasites from fecal samples.
The present invention overcomes the deficiencies of prior devices by providing a multi-filter apparatus for quantitatively separating ova and parasites from human and animal specimens and for concentrating the parasite samples. The multi-filter apparatus eliminates the need for separate filtration and separation steps by performing both steps at the same time. An additional advantage is that the use of toxic chemicals such as diethyl ether and ethyl acetate is eliminated. The multi-filter apparatus may be used a part of a specimen-processing kit.
The present invention also includes a method for processing human and animal specimens by mixing the specimen in a preservation solution, transferring the specimen-containing solution to a specimen receptacle and then fractionating the mixture in the multi-filter apparatus. During fractionation, the ova and parasites are concentrated in a collection receptacle. The concentrated ova and parasites are then conveniently separated from the supernatant and are ready for subsequent analysis.